HPLC-荧光检测法测定大鼠血浆中大黄酸的浓度及其药代动力学

Determination of Rhein in Plasma by HPLC-Fluorescence Detection and its Parmacokinetics in Rats

目的:建立测定大鼠血浆中大黄酸浓度的HPLC荧光检测方法,并对大黄酸在大鼠体内的药代动力学行为进行研究。方法:血浆样品经乙醚萃取后,用HPLC荧光法(HPLCFLD法)进行测定分析。色谱柱为C18Hypersil,ODS2,5μm,250mm×4.6mmID,流动相为0.5%的冰醋酸-乙腈-甲醇(27∶10∶60),检测激发波长440nm,发射波长520nm,同时测定大鼠单次灌胃大黄酸70mg/kg后血药浓度,并利用DAS软件拟合其药代动力学参数。结果:大黄酸的血药浓度在0.052～80μg/mL范围内线性关系良好,最低检测限为2ng/mL,以质控样品计算,在各浓度水平下,此法的回收率均大于85%,日间和日内精密度小于15%,符合生物样品分析要求。大鼠灌胃大黄酸70mg/kg后,血药浓度-时间曲线呈二室模型。主要药动学参数Tmax,Cmax,AUC(0-t),MRT,T1/2α,T1/2β分别为0.50±0.27h,54.64±11.60μg/mL,164.29±44.77μg/h·mL,4.03±0.46h,1.48±0.77h,3.68±1.42h。结论:该法操作简便、快速、灵敏,适用于对微量生物样品进行大批量测定。

AIM: To establish a RP-HPLC-fluorescence method for the determination of rhein in plasma and to study pharmacokinetics of rhein in rats. METHOD: Plasma samples were prepared based on a liquid-liquid extraction by aether. The extracted samples were analyzed by liquid chromatography. Rhein was eluted on a Hypersil-ODS2, 5 μm, 250 mm×4.6 mm I.D column, using a mobile phase of 0.5% acetic acid-acetonitrile-methanol (27∶10∶60). Rhein was detected on flurescenct-detector at λex 440 nm and λem 520 nm. The pharmacokinetics of rhein in rats after a single dose of 70 mg/kg was studied in this experiment. RESULT: Calibration curves were linear over 0.052～80.0 μg/mL (r 2 =0.999) and LOD was 2 ng/mL, LOQ was 0.052 μg/mL, the recoveries of rhein from plasma were larger than 85%, and RSDs of inter-day and intra-day assay were below 15%. After i.g administration of 70 mg/kg rhein, the pharmacokinetic parameters of T_ max , C_ max , AUC_ (0-t) , MRT, T_ 1/2α , T_ 1/2β were 0.5±0.27 h , 54.64±11.60 μg/mL, 164.29±44.27 μg/h·mL, 4.03±0.46 h, 1.48±0.77 h, 3.68±1.42 h, resepectively. CONCLUSION: A reliable HPLC-fluorescence assay determination of rhein in rat plasma was developed and this method can be applied to pharmacokinetic study.